Resistance biomarkers for hdac inhibitors

ABSTRACT

Provided herein are methods for identifying a cancer patient at risk for resistance to an HDAC inhibitor therapy, comprising obtaining a tumor sample from the cancer patient; detecting the presence of Testis-specific Y-encoded-like protein 5 (TSPYL5) expression in the sample; quantifying a level of the TSPYL5 expression in the sample, wherein a high level of the TSPYL5 expression, relative to a defined expression threshold of the TSPYL5, correlates with resistance to the HDAC inhibitor therapy; and applying the correlation to identify the cancer patient at risk for resistance to the HDAC inhibitor therapy. Also provided is a method for identifying a cancer patient with an increased likelihood of a positive clinical response to an HDAC inhibitor therapy comprising obtaining a tumor sample from the cancer patient; detecting the presence of Testis-specific Y-encoded-like protein 5 (TSPYL5) expression in said sample; quantifying a level of said TSPYL5 expression in said sample, wherein a low level of the TSPYL5 expression, relative to a defined expression threshold of the TSPYL5, identifies said cancer patient with an increased likelihood of a positive clinical response to said HDAC inhibitor therapy. Related methods and compositions are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/019,456 filed Sep. 5, 2013, now allowed, which claims priority toU.S. provisional application Ser. No. 61/698,341, filed Sep. 7, 2012;U.S. provisional application Ser. No. 61/726,464, filed Nov. 14, 2012;and U.S. provisional application Ser. No. 61/784,501, filed Mar. 14,2013, which are incorporated herein by reference in their entireties.

FIELD

The invention relates generally to the field of personalized medicineand, more specifically to the discovery that TSPYL5, encodingtestis-specific Y-like protein, serves as a tumor biomarker forresistance to cancer treatment with a histone deacetylase (HDAC)inhibitor.

BACKGROUND

Cancer is a major public health problem in the United States and in theworld. Currently, one in 4 deaths in the United States is due to cancer.Each year, the American Cancer Society estimates the numbers of newcancer cases and deaths expected in the United States in the currentyear and compiles the most recent data on cancer incidence, mortality,and survival based on incidence data from the National Cancer Institute,the Centers for Disease Control and Prevention, and the North AmericanAssociation of Central Cancer Registries and mortality data from theNational Center for Health Statistics. A total of 1,596,670 new cancercases and 571,950 deaths from cancer were projected to occur in theUnited States in 2011. Aging of the general population and developmentof new forms of cancer contribute to the problem.

Attempts have been made to identify genes or other markers that wouldeither predict response to treatment, or correlate with response totreatment. In 2009, the laboratory of Nicholas B. La Thangue publishedthe results of a genome-wide loss of function screen that identified arole for HR23B as a sensitivity determinant for HDAC inhibitor thatinduced apoptosis in cells (Fotheringham et al., Cancer Cell 15:57(2009). In a subsequent paper, the authors noted a frequent coincidencebetween HR23B expression and clinical response to HDAC inhibition (Khanet al., PNAS 107:6532 (2010).

Other studies described markers that correlate with sensitivity to HDACinhibitors in cells. Shao et al. (Int. J. Cancer 127:2199 (2010))compared 4 lines that are either sensitive or resistant to panobinostattreatment and found that inhibition of BCL2 sensitized resistant linesto panobinostat treatment. BCL2 blocks the pro-apoptotic activity ofBAX, and knockdown of BAX was found to diminish sensitivity topanobinostat treatment. These results were in line with previous studiesshowing that overexpression of BCL2 and BCL-xl blocked HDAC inhibitormediated apoptosis (Bolden et al., Nature Reviews Drug Discovery 5:769(2006), including apoptosis mediated by romidepsin (Peart et al., CancerResearch 63:4460 (2003). Later studies showed that romidepsin is able toinduce apoptosis in lymphomas overexpressing BCL2 with delayed kinetics,but not in cells overexpressing BCL-xl (Newbold et al, Mol. Cancer Ther.7:1066 (2008); WO/2010/047714). Peart et al. confirmed romidepsin as asubstrate for P-glycoprotein (P-gp), and showed that cellsoverexpressing P-gp are resistant to apoptosis induced by the drug. AlsoScala showed romidepsin to be a P-gp substrate (Scala et al., MolecularPharmacology 51:1024 (1997) and a substrate for Multidrug ResistanceAssociated Protein 1 (MRP1), but the major mechanism of acquiredresistance to romidepsin in cells appears to be up-regulation of P-gp(Xiao et al., J Pharmacol and Exp Ther 313:268 (2005)). In spite of thecorrelation between P-gp expression and romidepsin sensitivity that isobserved in cell culture assays, no association exists between P-gpexpression and clinical response (Bates et al., Br J Haematol 148:256(2010).

Various laboratories have tried to establish gene expression signaturesthat correlate with response to treatment to HDAC inhibitors (Stimson etal., Cancer Lett 280:177 (2009)). However, these signatures vary fromstudy to study and are most likely unique to the tumor type studied andthe HDAC inhibitor used. For example, Yuka Sasakawa and colleagues triedto identify markers that predict sensitivity to romidepsin (Sasakawa etal., Biochem Pharmacol 69:603 (2005)). This study compared expressionprofiles of sensitive and resistant to romidepsin tumors and identifiedcaspase 9 and MKP-1 genes as marker genes to predict sensitivity toromidpsin treatment. However, the validity of these markers is likely tobe limited to these specific studies.

Between 2,000 and 3,000 new cases of cutaneous T-cell lymphoma (CTCL)occur in the United States each year, with mycosis fungoides (MF) andthe Sézary syndrome (SS) being the predominant subtypes. Romidepsinactivity in T-cell lymphomas was observed in phase I and II trialsconducted by the National Cancer Institute (NCI) in patients with bothMF and SS. (Piekarz et al., Blood 103: 4636 (2004); Sandor et al., ClinCancer Res 8:718 (2002); Marshall et al., J Exp Ther Oncol 2:325 (2002);Piekarz et al., Blood 98:2865 (2001); Piekarz et al., J. ClinicalOncology 27 (32):5410 (2009)). Romidepsin was shown in a phase IIclinical trial to have single-agent clinical activity with significantand durable responses in patients with cutaneous T-cell lymphoma (CTCL)(Piekarz et al., J. Clinical Oncology 27 (32):5410 (2009)). Romidepsinhas also been shown to have significant and sustainable single-agentactivity and an acceptable safety profile for treatment of refractoryCTCL (Whittaker et al. J Clin Oncol 28:4485-4491 (2010)).

Little is known about TSPYL5, which encodesTestis-specificY-encoded-like protein 5. It contains a nucleasomeassembly protein domain (NAP-domain) that acts as histone chaperone.TSPYL5 has been shown to be involved in cell growth and resistance toradiation in A549 cells (Kim et al., Biochem and Biophys Res Comm392:448 (2010). It is a target of epigenetic silencing in gastriccancers (Jung et al., Lab Invest 88:153 (2008), and glioma (Kim et al.,Cancer Res 66:7490 (2006)) and is thought to mediate some of itsfunction by suppressing p53 activity via physical interaction with USP7(Epping et al., Nature Cell Biol 13:102 (2011). There is no knownconnection between the levels of TSPYL5 and sensitivity to treatmentwith romidepsin or other HDAC inhibitors.

Currently, patients receiving treatment with romidepsin are not selectedfor treatment based on the expression of predictive markers. To improveclinical outcomes, a need exists to identify biomarkers that allowselecting cancer patients that are more likely to respond positively toHDAC inhibitor therapy while deselecting cancer patients that are likelyto be resistant to HDAC inhibitor therapy.

SUMMARY

In one aspect of the invention, a method is provided for identifying acancer patient at risk for resistance to an HDAC inhibitor therapy,comprising obtaining a tumor sample from the cancer patient; detectingthe presence of Testis-specific Y-encoded-like protein 5 (TSPYL5)expression in the sample; quantifying a level of the TSPYL5 expressionin the sample, wherein a high level of the TSPYL5 expression, relativeto a defined expression threshold of the TSPYL5, correlates withresistance to the HDAC inhibitor therapy; and applying the correlationto identify the cancer patient at risk for resistance to the HDACinhibitor therapy.

Also provided is a method for identifying a cancer patient with anincreased likelihood of a positive clinical response to an HDACinhibitor therapy comprising obtaining a tumor sample from the cancerpatient; detecting the presence of Testis-specific Y-encoded-likeprotein 5 (TSPYL5) expression in said sample; quantifying a level ofsaid TSPYL5 expression in said sample, wherein a low level of the TSPYL5expression, relative to a defined expression threshold of the TSPYL5,identifies said cancer patient with an increased likelihood of apositive clinical response to said HDAC inhibitor therapy.

In some embodiments, the methods provided herein further comprisecommunicating the identification of the cancer patient to a health careprovider. In additional embodiments, the communication to a health careprovider informs a subsequent treatment selection for the cancerpatient. In certain embodiments the treatment selection involves eitherselecting or deselecting the cancer patient for HDAC inhibitor therapy.

In additional embodiments, the methods provided herein further compriseadministering a therapeutically effective amount of the HDAC inhibitorto the selected patient. In further embodiments, the HDAC inhibitor isselected from the group consisting of romidepsin, panobinostat,vorinostat and entinostat. In a particular embodiment, the HDACinhibitor is romidepsin.

In further embodiments, the methods provided herein further compriseinitiating HDAC inhibitor therapy. In further embodiments, the HDACinhibitor therapy comprises an HDAC inhibitor selected from the groupconsisting of romidepsin, panobinostat, vorinostat and entinostat. In aparticular embodiment, the HDAC inhibitor is romidepsin.

Also provided are embodiments where the HDAC inhibitor is selected fromthe group consisting of romidepsin, panobinostat, vorinostat andentinostat. In certain embodiments, the HDAC inhibitor is romidepsin.

In yet further embodiments, the level of the TSPYL5 expression isdetermined by measuring the amount of TSPYL5 protein using animmunoassay, for example, an immune-polymerase chain reaction(immuno-PCR).

Also provided are kits comprising a container filled with an HDACinhibitor, reagents for determining the level of the TSPYL5 gene orprotein in a tumor sample, and instructions for determining the level ofexpression of TSPYL5 gene or protein in a tumor sample of a patienthaving cancer.

The present embodiments can be understood more fully by reference to thedetailed description and examples, which are intended to exemplifynon-limiting embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a sample data plot showing cell growth at variousconcentrations of romidepsin after 6 h and 72 h drug treatment. Percentgrowth is plotted against drug concentration. Cell growth for bothtreatments was measured after 72 h using CellTiter-Glo®. Measurementswere normalised to numbers of cells at the beginning of the experiment(d0) as measured by CellTiter-Glo®.

FIG. 2A shows romidepsin S-values in different cell lines as a whiskerplot. Sensitivity of cell lines to romidepsin treatment was determinedusing 6 h drug treatment and is expressed using the S value. The lowerthe value, the more sensitive the cell line is to romidepsin treatment.Cell lines are grouped according to their tissue of origin, and for eachcell line, the romidpsin S value is plotted against the tissue oforigin. The sensitivity of the cell lines varies greatly, with S valuesranging from 4-2000. FIG. 2B demonstrates the sensitivity distributionin various cell lines based on the S-Value using a box plot showing thesmallest observation (sample minimum), lower quartile (Q1), median (Q2),upper quartile (Q3), and largest observation (sample maximum).Sensitivity of cell lines to romidepsin treatment was determined using 6h drug treatment and is expressed using the S value. The lower thevalue, the more sensitive the cell line is to romidepsin treatment. Celllines are grouped according to their tissue of origin, and cell lineorigin is plotted against the romidepsin S value. Sensitivity ofdifferent types of cancer cell lines to romidepsin varies greatly. Asshown here, blood cell lines show the greatest sensitivity.

FIGS. 3A-3B depict the correlation between TSPYL5 gene expression andS-values in a cell line screen. Gene expression of TSPYL5 was quantifiedin a set of cell lines using an Affymetrix gene profiling array andplotted against the sensitivity of the cell line to either romidepsin(FIG. 3A) or panobinostat (FIG. 3B) as measured by their S value. Thelower the S value, the higher the sensitivity to drug treatment. Cellslines with more than baseline expression of TSPYL5 have high S values,that is, they are resistant to treatment with either romidepsin or withpanobinostat.

FIG. 4 depicts the correlation between TSPYL5 gene expression and theIC₅₀ value of romidepsin in primary patient tumors explants. Romidepsinresistance of primary tumors correlates with TSPYL5 expression. Humanprimary tumors were propagated in mice. For IC₅₀ measurements, tumorswere dissociated and grown in a clonogenic assay in the presence ofdifferent concentrations of romidepsin. The graph plots TSPYL5expression of the primary tumors against romidepsin IC₅₀ as determinedin the clonogenic assays. Primary tumors with high TSPYL5 expressionhave relatively high IC₅₀s.

FIGS. 5A-5C depict the effect of knockdown of TSPYL5 expression onsensitivity of SKOV-3 cells to romidepsin using shRNA, as measured bythe effect of romidepsin treatment on IC₅₀ (FIG. 5A), growth rate (GR)(FIG. 5B), and S value (FIG. 5C). Sensitivity of TSPYL5 knockdown cellsto romidepsin is increased as measured by IC₅₀, S value and growthinhibition. SKOV3 cells expressing either TSPYL5 shRNA (SKOV3 TSPYL5 KD)or non-silencing control shRNA (SKOV3 NS) were created using lentiviralinfection. Knockdown of TSPYL5 in SKOV3 TSPYL5 KD cells was verifiedusing western blot (data not shown) and quantitative PCR. Expression ofTSPYL5 in these cells was reduced by 70% (data not shown) Afterselection of stable pools with puromycen, cells were treated withvarying concentrations of romidepsin for determination of IC₅₀, GI and Svalue. The data shown are means and standard deviations from 5independent experiments. FIG. 5A: Romidepsin IC₅₀s for SKOV3 TSPYL5 KDcells and SKOV3 NS cells. FIG. 5B: Romidepsin GI for SKOV3 TSPYL5 KDcells and SKOV3 NS cells. FIG. 5C: Romidepsin S value for SKOV3 TSPYL5KD cells and SKOV3 NS cells. Numbers in white are means obtained from 5experiments. The asterisks describe values levels of statisticalsignificance, with 2 asterisks depicting p-values between 0.01 and0.001.

FIG. 6 provides a table comparing the inhibitory activity for HistoneDeacetylases 1 through 9 of 4 commonly used HDAC inhibitors. HDACs 1, 2,3 and 8) are known as class 1 HDACs, while HDACs 4, 5, 6, 7 and 9 areknown as class 2 HDACs. The table shows Ki in nM, and relativeactivities compared to HDAC 1 (NI: no inhibition). All 4 HDAC inhibitorsinhibit the class 1 HDACs 1 and 2. MS-275 and romidepsin are moreselective than the hydroxamic acids panobinostat and SAHA, which inhibitthe class 2 HDAC 6 in addition to inhibiting class 1 HDACs 1, 2 and 3,and, to a lesser degree, HDAC 8. Based on data from Bradner et al., NatChem Biol 6:238 (2010).

FIGS. 7A-7C depict the effect of knockdown of TSPYL5 expression onsensitivity of SKOV-3 cells to panobinostat using shRNA, as measured bythe effect of panobionstat treatment on IC50 (FIG. 7A), growth rate (GR)(FIG. 7B), and S value (FIG. 7C). Sensitivity of TSPYL5 knockdown cellsto panobinostat is increased as measured by S value and growth rate.SKOV3 cells expressing either TSPYL5 shRNA (SKOV3 TSPYL5 KD) ornon-silencing control shRNA (SKOV3 NS) were created using lentiviralinfection. Knockdown of TSPYL5 in SKOV3 TSPYL5 KD cells was verifiedusing western blot (data not shown) and quantitative PCR. Expression ofTSPYL5 in these cells was reduced by 70%. After selection of stablepools with puromycen, cells were treated with varying concentrations ofpanobinostat for determination of IC₅₀, GR and S value. The data shownare means and standard deviations from 5 independent experiments. FIG.7A: panobinostat IC₅₀s for SKOV3 TSPYL5 KD cells and SKOV3 NS cells.FIG. 7B: panobinostat GR for SKOV3 TSPYL5 KD cells and SKOV3 NS cells.FIG. 7C: panobinostat S value for SKOV3 TSPYL5 KD cells and SKOV3 NScells. Numbers in white are means obtained from 5 experiments. Theasterisks describe values levels of statistical significance, with 1asterisks depicting p-values between 0.01 and 0.05.

FIGS. 8A-8C depict the effect of knockdown of TSPYL5 expression onsensitivity of HDF cells to romidepsin using shRNA, as measured by theeffect of romidepsin treatment on IC₅₀ (FIG. 8A), growth rate (GR) (FIG.8B), and S value (FIG. 8C). Sensitivity of TSPYL5 knockdown cells toromidepsin is increased as measured by S value and growth rate. HDFcells expressing either TSPYL5 shRNA (HDF TSPYL5 KD) or non-silencingcontrol shRNA (HDF NS) were created using lentiviral infection.Knockdown of TSPYL5 in HDF TSPYL5 KD cells was verified using westernblot and quantitative PCR. Expression of TSPYL5 in these cells wasreduced by >70%. After selection of stable pools with puromycen, cellswere treated with varying concentrations of romidepsin for determinationof IC₅₀, GR and S value. The data shown are means and standarddeviations from 8 independent experiments. FIG. 8A: Romidepsin IC₅₀s forHDF TSPYL5 KD cells and HDF NS cells. FIG. 8B: Romidepsin GR for HDFTSPYL5 KD cells and HDF NS cells. FIG. 8C: Romidepsin S value for HDFTSPYL5 KD cells and HDF NS cells. Numbers in white are means obtainedfrom 8 experiments. The asterisks describe values levels of statisticalsignificance, with 2 asterisks depicting p-values between 0.001 and0.01.

DETAILED DESCRIPTION Definitions

As used in the specification and the accompanying claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents, unless the context clearly dictatesotherwise.

As used herein, and unless otherwise specified, the term “about” or“approximately” means an acceptable error for a particular value asdetermined by one of ordinary skill in the art, which depends in part onhow the value is measured or determined. In certain embodiments, theterm “about” or “approximately” means within 1, 2, 3, or 4 standarddeviations. In certain embodiments, the term “about” or “approximately”means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, or 0.05% of a given value or range.

As used herein, and unless otherwise specified, the term “HDAC inhibitortherapy” refers to the administration of an HDAC inhibitor to a patientin order to effect the treatment, eradication or amelioration of acondition, disorder, or disease, or of one or more symptoms associatedwith the a condition, disorder, or disease. In certain embodiments, theadministration can be aimed to minimize the spread or worsening of thedisease or disorder resulting from the administration of the HDACinhibitor to a subject with such a disease or disorder. In someembodiments, the term refers to the administration of a compound ordosage form provided herein, with or without one or more additionalactive agent(s), after the diagnosis or the onset of symptoms of thedisease. In some embodiments, the term may encompass prevention.

As used herein, and unless otherwise specified, the term“therapeutically effective amount” in connection with the HDAC inhibitorrefers to that amount of the compound being administered sufficient toslow the development of or alleviate to some extent one or more of thesymptoms of the condition or disorder being treated, for example cancer,or slowing or halting further progression or worsening of thosesymptoms, in a subject at risk for cancer. The effective amount of theHDAC inhibitor, for example in a pharmaceutical composition, may be at alevel that will exercise the desired effect; for example, about 0.005mg/m² to 100 mg/m², about 0.05 mg/m² to 90 mg/m², about 0.5 mg/m² to 80mg/m², about 1.0 mg/m² to 70 mg/m², about 2.0 mg/m² to 60 mg/m², about3.0 mg/m² to 50 mg/m², about 4.0 mg/m² to 40 mg/m², about 5.0 mg/m² to30 mg/m², about 10.0 mg/m² to 20 mg/m², about 11.0 mg/m² to 19 mg/m²,about 12.0 mg/m² to 18.0 mg/m², about 13.0 mg/m² to 17.0 mg/m², about14.0 mg/m² to 16.0 mg/m², about 14.5 mg/m² to 15.5 mg/m², about 14.6mg/m² to 15.4 mg/m², about 14.7 mg/m² to 15.3 mg/m², about 14.8 mg/m² to15.2 mg/m² of a subject's body weight, in unit dosage for both oral andparenteral administration. As will be apparent to those skilled in theart, it is to be expected that the effective amount of an HDAC inhibitordisclosed herein may vary depending on the severity of the indicationbeing treated.

A “biological marker” or “biomarker” is a substance, the change and/orthe detection of which indicates a particular biological state, such as,for example, the resistance of a disease, for example, cancer, to agiven treatment, for example, HDAC inhibitor therapy.

An “increased likelihood” in reference to a positive clinical responseis intended to mean that a cancer patient has a higher likelihood torespond to HDAC inhibitor therapy compared to the average likelihood ofresponsiveness to HDAC inhibitor therapy calculated from a random poolof cancer patients.

The term “responsiveness” or “responsive” when used in reference to atreatment refer to the degree of effectiveness of the treatment inlessening or decreasing the symptoms of a disease, e.g., cancer.

The term “positive clinical response” when used in reference to a HDACinhibitor therapy refers to a lessening or decrease of one or more ofthe symptoms of the disease treated.

The term “expressed” or “expression” refers to the transcription from agene to produce an RNA nucleic acid molecule, e.g., mRNA, at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from an RNA molecule to give a protein, apolypeptide, or a portion thereof.

The terms “expression threshold,” and “defined expression threshold” areused interchangeably and refer to the level of a gene or protein abovewhich the gene or gene product serves as a predictive marker for patientresistance to HDAC inhibitor therapy. The expression threshold is arelative level and is established by quantifying the expression ofTSPYL5 in the tumor cells of a number of patients with similar tumors.The expression level of TSPYL5 found in the tumor cells of the patientswith the lowest expression level is defined as the expression threshold.Patients are considered likely to respond to treatment with an HDACinhibitor if their tumor has expression levels close to that threshold,while patients are considered likely to be resistant to treatment withan HDAC inhibitor if their tumor has expression levels higher than thatthreshold. The threshold can be defined experimentally from clinicalstudies. The expression threshold can be selected either for maximumsensitivity, or for maximum selectivity, or for minimum error. Thedetermination of the expression threshold is well within the knowledgeof those skilled in the art.

A “low” level of TSPYL5 expression is a level of expression at or belowa predetermined expression threshold. A “high” level of a TSPYL5expression is a level of TSPYL5 gene expression above a predeterminedexpression threshold.

It is understood that the genes and/or proteins described herein areinclusive of allelic variant isoforms, synthetic nucleic acids and/orproteins, nucleic acid and/or proteins isolated from tissue and cells,and modified forms thereof. It is also understood that the genes and/orproteins described herein are also known to exist in various forms,including variants and mutants, and are contemplated herein. The genesand/or proteins described herein further include nucleic acid sequencesand/or amino acid sequences having at least 65% identity with the geneor protein to be detected and are included within embodiments describedherein.

A used herein, the term “subject” or “patient” refers generally to amammal. In particular embodiments, the term refers to a cancer patientthat has been diagnosed as having cancer.

As used herein, and unless otherwise specified, the term “unit dose”when used in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for humans, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluents or excipients, i.e., carrier, or vehicle.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. The term “tumor sample” refers to abiological sample useful for detection of TSPYL5 comprising tumor cellsand includes, without limitation, biopsies, tissues, blood, cells,secretions, cerebrospinal fluid, bile, lymph fluid, urine and faeces, ortissue which has been removed from organs, such as, for example, breast,lung, intestine, skin, cervix, prostate, and stomach. For example, atumor sample can comprise a region of functionally related cells oradjacent cells as well as circulating tumor cells isolated from blood.In one example, a tumor sample includes blood obtained from a cancerpatient, such as whole blood or serum.

As used herein, and unless otherwise specified, the term “biologicalsample,” generally refers to a sample obtained from a biologicalsubject, including a sample of biological tissue or fluid origin,obtained, reached, or collected in vivo or in situ. A biological samplealso includes samples from a region of a biological subject containingprecancerous or cancer cells or tissues. Such samples can be, but arenot limited to, organs, tissues, fractions and cells isolated from amammal. Exemplary biological samples include but are not limited to celllysate, a cell culture, a cell line, a tissue, oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a serum sample, a urine sample, a skin sample, and thelike. Preferred biological samples include but are not limited to wholeblood, partially purified blood, PBMCs, tissue biopsies, and the like.

As used herein, and unless otherwise specified, the terms “cancer” and“cancerous” refer to or describe a group of diseases which arecharacterized by uncontrolled growth and spread of abnormal cells.Cancers include, but are not limited to, carcinomas, sarcomas,leukemias, lymphomas and the like. In certain embodiments, cancer is ahematological malignancy. In certain embodiments, cancer is a solidtumor.

In certain embodiments the present disclosure relates to treatment ofhematological malignancies. Manifestations of hematological malignanciesinclude circulating malignant cells and malignant masses. Hematologicalmalignancies are types of cancers that affect the blood, bone marrow,and/or lymph nodes. Hematological malignancies that may be treated usingromidepsin include, but are not limited to: acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma(CTCL), peripheral T-cell lymphoma (PTCL), multiple myeloma, andmyelodysplastic syndromes. In certain embodiments, romidepsin is used totreat multiple myeloma. In certain particular embodiments, the cancer isrelapsed and/or refractory multiple myeloma. In other embodiments,romidepsin is used to treat chromic lymphocytic leukemia (CLL). Incertain particular embodiments, the cancer is relapsed and/or refractoryCLL. In other embodiments, romidepsin is used to treat chromicmyelogenous leukemia (CML). In certain embodiments, romidepsin is usedto treat acute lymphoblastic leukemia (ALL). In certain embodiments,romidepsin is used to treat acute myelogenous leukemia (AML). In certainembodiments, the cancer is cutaneous T-cell lymphoma (CTCL). In otherembodiments, the cancer is peripheral T-cell lymphoma (PTCL). In certainembodiments, the cancer is a myelodysplastic syndrome.

In some embodiments of the present disclosure, cancers treated include,but are not limited to, leukemias and lymphomas such as cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma, lymphomas associated withhuman T-cell lymphotropic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), B-cell lymphomas, acute lymphocytic leukemia,acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease,non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndromes.

In some such embodiments the disclosure relates to treatment of solidtumors such as lung, breast, colon, liver, pancreas, renal, prostate,ovarian, and/or brain. In some embodiments, the disclosure relates totreatment of pancreatic cancer. In some embodiments, the disclosurerelates to treatment of renal cancer. In some embodiments, thedisclosure relates to treatment of prostate cancer. In some embodiments,the disclosure relates to treatment of sarcomas. In some embodiments,the disclosure relates to treatment of soft tissue sarcomas.

In some embodiments, cancers that can be treated are solid cancers thatinclude, but are not limited to, mesothelioma, common solid tumors ofadults such as head and neck cancers (e.g., oral, laryngeal andesophageal), genitourinary cancers (e.g., prostate, bladder, renal,uterine, ovarian, testicular, rectal and colon), melanoma and other skincancers, stomach cancer, brain tumors, liver cancer and thyroid cancer,and/or childhood solid tumors such as brain tumors, neuroblastoma,retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas.).In certain embodiments, the cancer is melanoma. In other embodiments,the cancer is gastric cancer. In some embodiments, the disclosurerelates to treatment of solid tumors.

Cancers that may be treated using the methods provided herein, includingcombination therapy, include but not limited to, colon cancer, lungcancer, bone cancer, pancreatic cancer, stomach cancer, esophagealcancer, skin cancer, brain cancer, liver cancer, ovarian cancer,cervical cancer, uterine cancer, testicular cancer, prostate cancer,bladder cancer, kidney cancer, and neuroendocrine cancer.

In certain embodiments, cancer is pancreatic cancer. In certainembodiments, cancer is prostate cancer. In certain specific embodiments,the prostate cancer is hormone refractory prostate cancer.

In some particular embodiments, provided are methods to treat leukemias.In some embodiments, leukemia is chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute lymphocytic leukemia, acute myelogenousleukemia, or adult T cell leukemia/lymphoma.

In some embodiments, provided are methods of treating lymphomas. In someembodiments, lymphoma is Hodgkin's or non-Hodgkin's (e.g., T-celllymphomas such as peripheral T-cell lymphoma, cutaneous T-cell lymphoma,etc.) lymphoma.

In some embodiments, the disclosure relates to the treatment of multiplemyeloma and/or myelodysplastic syndromes.

As used herein, and unless otherwise specified, the term “resistance”refers to the condition when a disease does not respond to the treatmentof a drug or drugs. Drug resistance can be either intrinsic, which meansthe disease has never been responsive to the drug or drugs, or it can beacquired, which means the disease ceases responding to a drug or drugsthat the disease had previously responded to. In certain embodiments,drug resistance is intrinsic. In certain embodiments, the drugresistance is acquired.

As used herein, and unless otherwise specified, the term “histonedeacetylase inhibitor” or “HDAC inhibitor” refers to a compound thatmodulates protein acetylation by blocking zinc-dependent histonedeacetylases involved in removing acetyl groups from lysine residues.HDAC inhibitors can be separated into several structurally distinctclasses: short-chain fatty acids (i.e., valproic acid), hydroxamic acids(i.e., vorinostat, TSA, tubacin, and PCI-24781), benzamides (i.e.,entinostat), cyclic tetrapeptides (i.e., romidepsin), and electrophilicketones. For example, the HDAC inhibitors romidepsin, panobinostat,vorinostat (SAHA) and entinostat (MS-275) inhibit the class 1 HDACs 1and 2. Entinostat and romidepsin are more selective than the hydroxamicacids panobinostat and SAHA, which inhibit the class 2 HDAC 6 inaddition to inhibiting class 1 HDACs 1, 2 and 3, and, to a lesserdegree, HDAC 8. Based on data from Bradner et al., Nat Chem Biol 6:238(2010).

As used herein, and unless otherwise specified, the terms “determining,”“measuring,” “evaluating,” “assessing,” and “assaying,” generally referto any form of measurement, and include determining if an element ispresent or not. These terms include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute. The phrase“assessing the presence of” can include determining the amount ofsomething present, as well as determining whether it is present orabsent.

As used herein, and unless otherwise specified, the phrase “assessingthe activity of an agent,” encompasses the assessment of the “presence”of the treatment by the agent, e.g., whether the patient has beentreated by or administered the agent compound. The phrase alsoencompasses the assessment of the “extent” of the treatment, e.g., dosesand length of treatment determined in quantitative terms. The phrasealso encompasses assessing the effect of the agent, e.g., response orresults of the treatment.

As used herein, and unless otherwise specified, the terms “isolated” and“purified” generally describes a composition of matter that has beenremoved from its native environment (e.g., the natural environment if itis naturally occurring), and thus is altered by the hand of man from itsnatural state. An isolated protein or nucleic acid is distinct from theway it exists in nature.

The term “polypeptide,” “protein,” or “peptide,” as used hereininterchangeably, refers to a polymer of two or more amino acids in aserial array, linked through one or more peptide bond(s). The termencompasses proteins, protein fragments, protein analogues,oligopeptides, peptides, and peptide mimics. The amino acids of apolypeptide, protein, or peptide can be naturally occurring amino acidsor synthetic amino acids (e.g., mimics of naturally occurring aminoacids). A polypeptide, protein, or peptide can be made synthetically orpurified from a biological sample. The term also encompasses modifiedpolypeptides, proteins, and peptides, e.g., a depsipeptide,glycopolypeptide, glycoprotein, or glycopeptide; or a lipopolypeptide,lipoprotein, or lipopeptide.

The term “antibody” refers to a polypeptide that specifically binds anepitope (e.g., an antigen). The term “antibody” is used herein in thebroadest sense and covers fully assembled antibodies, antibody fragmentswhich retain the ability to specifically bind to an antigen (e.g., Fab,F(ab′)₂, Fv, and other fragments), single chain antibodies, diabodies,antibody chimeras, hybrid antibodies, bispecific antibodies, andhumanized antibodies. The term “antibody” also covers both polyclonaland monoclonal antibodies.

As used herein, and unless otherwise specified, the term “label” or a“detectable moiety” in reference to a protein, generally refers to acomposition that, when linked with a protein, renders the proteindetectable, for example, by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. Exemplary labels include but are notlimited to radioactive isotopes, magnetic beads, metallic beads,colloidal particles, fluorescent dyes, enzymes, biotin, digoxigenin,haptens, and the like. A “labeled protein or oligopolypeptide probe” isgenerally one that is bound, either covalently, through a linker or achemical bond, or noncovalently, through ionic bonds, van der Waalsforces, electrostatic attractions, hydrophobic interactions, or hydrogenbonds, to a label such that the presence of the protein or probe can bedetected by detecting the presence of the label bound to the protein orprobe.

The term “probe” as used herein, refers to a capture agent, for example,a nucleic acid sequence, that is directed to a specific target DNA ormRNA biomarker sequence. Accordingly, each probe of a probe set has arespective target DNA or mRNA biomarker. A probe/target DNA or mRNAduplex is a structure formed by hybridizing a probe to its target DNA ormRNA biomarker.

The term “nucleic acid probe” or “oligonucleotide probe” refers to anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence, such as the mRNA biomarkers provided herein,through one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (e.g., A, G, C, or T) ormodified bases (7-deazaguanosine, inosine, etc.). In addition, the basesin a probe may be joined by a linkage other than a phosphodiester bond,so long as it does not substantially interfere with hybridization. Itwill be understood by one of skill in the art that a probe may bind atarget sequence lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridization conditions. Theprobes are preferably directly labeled with, for example, isotopes,chromophores, lumiphores, or chromogens, or indirectly labeled withbiotin to which a streptavidin complex may later bind. By assaying forthe presence or absence of the probe, one can detect the presence orabsence of a target DNA or mRNA biomarker of interest.

The term “stringent assay conditions” refers to conditions that arecompatible to produce binding pairs of nucleic acids, e.g., probes andtarget DNA or mRNAs, of sufficient complementarity to provide for thedesired level of specificity in the assay while being generallyincompatible to the formation of binding pairs between binding membersof insufficient complementarity to provide for the desired specificity.The term “stringent assay conditions” generally refers to thecombination of hybridization and wash conditions.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom the administration site of one organ, or portion of the body, toanother organ, or portion of the body, or in an in vitro assay system.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to a subjectto whom it is administered. Nor should an acceptable carrier alter thespecific activity of the subject compounds.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases known in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

The term “prodrug” means a derivative of a compound that can hydrolyze,oxidize, or otherwise react under biological conditions (in vitro or invivo) to provide the compound. Examples of prodrugs include, but are notlimited to, derivatives of romidepsin, its reduced, oxidized, andoligomerized forms. Prodrugs can typically be prepared using well-knownmethods, such as those described in 1 Burger's Medicinal Chemistry andDrug Discovery, 172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995),and Design of Prodrugs (H. Bundgaard ed., Elsevier, New York 1985).

The practice of the embodiments provided herein will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, and immunology, which are within the skill of thoseworking in the art. Such techniques are explained fully in theliterature. Examples of particularly suitable texts for consultationinclude the following: Sambrook et al., Molecular Cloning; A LaboratoryManual (2d ed.), 1989; Glover, ed. DNA Cloning, Volumes I and II, 1985;Gait, ed., Oligonucleotide Synthesis, 1984; Hames & Higgins, eds.Nucleic Acid Hybridization, 1984; Hames &. Higgins, eds., Transcriptionand Translation, 1984; Freshney, ed., Animal Cell Culture, 1986;Immobilized Cells and Enzymes, IRL Press, 1986; Immunochemical Methodsin Cell and Molecular Biology (Academic Press, London); Scopes, ProteinPurification: Principles and Practice (2d ed.; Springer Verlag, N.Y.),1987; and Weir and Blackwell, eds. Handbook of Experimental Immunology,Volumes I-IV, 1986.

Romidepsin

Romidepsin is a natural product which was isolated from Chromobacteriumviolaceum by Fujisawa Pharmaceuticals (Published Japanese PatentApplication Hei 7 (1995)-64872; and U.S. Pat. No. 4,977,138, issued Dec.11, 1990, each of which is incorporated herein by reference). Variouspreparations and purifications of romidepsin are described in PCTPublication WO 2002/20817, which is incorporated herein by reference.Solid forms of romidpesin are described in U.S. Pat. Nos. 7,608,280 and7,611,724, a method of manufacturing romidepsin is described in US2010/0093610 and US 2009/0209616, and a romidpesin formulation isdescribed in US2012/0046442, and each of the aforementioned isincorporated herein by reference in its entirety.

Romidepsin is a bicyclic depsipeptide consisting of four amino acidresidues (D-valine, D-cysteine, dehydrobutyrine, and L-valine) and anovel acid (3-hydroxy-7-mercapto-4-heptenoic acid), which contains bothamide and ester bonds. Romidepsin can be obtained from C. violaceumusing fermentation. It can also be prepared by synthetic orsemi-synthetic means. The total synthesis of romidepsin reported by Kahnet al. (J. Am. Chem. Soc. 118:7237-7238, 1996) involves 14 steps andyields romidepsin in 18% overall yield. The structure of romidepsin isshown below (formula I):

Romidepsin has been shown to have antimicrobial, immunosuppressive, andanti-tumor activities. Romidepsin is sold under the tradename Istodax®and is approved in the United States for the treatment of cutaneousT-cell lymphoma (CTCL) in patients who have received at least one priorsystemic therapy, and for the treatment of peripheral T-cell lymphoma(PTCL) in patients who have received at least one prior therapy. It iswas tested for multiple myeloma and solid tumors (e.g., prostate cancer,pancreatic cancer, etc.) and is thought to act by selectively inhibitingdeacetylases (e.g., histone deacetylase, tubulin deacetylase) (Nakajimaet al., Exp Cell Res 241:126-133, 1998). One mode of action ofromidepsin involves the inhibition of one or more classes of histonedeacetylases (HDAC).

Exemplary forms of romidepsin include, but are not limited to, salts,esters, prodrugs, isomers, stereoisomers (e.g., enantiomers,diastereomers), tautomers, protected forms, reduced forms, oxidizedforms, derivatives, and combinations thereof, with the desired activity(e.g., deacetylase inhibitory activity, aggressive inhibition,cytotoxicity). In certain embodiments, romidepsin is a pharmaceuticalgrade material and meets the standards of the U.S. Pharmacopoeia,Japanese Pharmacopoeia, or European Pharmacopoeia. In certainembodiments, the romidepsin is at least 95%, at least 98%, at least 99%,at least 99.9%, or at least 99.95% pure. In certain embodiments, theromidepsin is at least 95%, at least 98%, at least 99%, at least 99.9%,or at least 99.95% monomeric. In certain embodiments, no impurities aredetectable in the romidepsin materials (e.g., oxidized material, reducedmaterial, dimerized or oligomerized material, side products, etc.).Romidepsin typically includes less than 1.0%, less than 0.5%, less than0.2%, or less than 0.1% of total other unknowns. The purity ofromidepsin may be assessed by appearance, HPLC, specific rotation, NMRspectroscopy, IR spectroscopy, UV/Visible spectroscopy, powder x-raydiffraction (XRPD) analysis, elemental analysis, LC-mass spectroscopy,or mass spectroscopy.

In one embodiment, romidepsin is present in a derivative form.

In one embodiment, the derivative of romidepsin is of the formula (II):

wherein

n is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

p and q are independently 1 or 2;

X is 0, NH, or NR₈;

R₁, R₂, and R₃ are independently hydrogen, unsubstituted or substituted,branched or unbranched, cyclic or acyclic aliphatic; unsubstituted orsubstituted, branched or unbranched, cyclic or acyclic heteroaliphatic;unsubstituted or substituted aryl; or unsubstituted or substitutedheteroaryl; and R₄, R₅, R₆, R₇ and R₈ are independently hydrogen, orsubstituted or unsubstituted, branched or unbranched, cyclic or acyclicaliphatic; and pharmaceutically acceptable forms thereof.

In one embodiment, m is 1, n is 1, p is 1, q is 1, X is 0, R₁, R₂, andR₃ are unsubstituted or substituted, branched or unbranched acyclicaliphatic. In one embodiment, R₄, R₅, R₆ and R₇ are all hydrogeIn oneembodiment, the derivative of romidepsin is of the formula (III):

wherein:

m is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

q is 2 or 3;

X is 0, NH, or NR₈;

Y is ORB, or 5 R₈;

R₂ and R₃ are independently hydrogen, unsubstituted or substituted,branched or unbranched, cyclic or acyclic aliphatic, unsubstituted orsubstituted, branched or unbranched, cyclic or acyclic heteroaliphatic,unsubstituted or substituted aryl or unsubstituted or substitutedheteroaryl;

R₄, R₅, R₆, R₇ and R₈ are independently selected from hydrogen orsubstituted or unsubstituted, branched or unbranched, cyclic or acyclicaliphatic, and pharmaceutically acceptable forms thereof.

In one embodiment, m is 1, n is 1, q is 2, X is NH and R₂ and R₃ areunsubstituted or substituted, branched or unbranched, acyclic aliphatic.In one embodiment, R₄, R₅, R₆ and R₇ are all hydrogen.

In one embodiment, the derivative of romidepsin is of the formula (IV):

wherein:

A is a moiety that is cleaved under physiological conditions to yield athiol group and includes, for example, an aliphatic or aromatic acylmoiety (to form a thioester bond), an aliphatic or aromatic thioxy (toform a disulfide bond), or the like, and pharmaceutically acceptableforms thereof. Such aliphatic or aromatic groups can include asubstituted or unsubstituted, branched or unbranched, cyclic or acyclicaliphatic group, a substituted or unsubstituted aromatic group, asubstituted or unsubstituted heteroaromatic group, or a substituted orunsubstituted heterocyclic group. A can be, for example, —COR₁,—SC(═0)-0-R₁, or —SR₂;

R₁ is independently hydrogen, substituted or unsubstituted amino,substituted or unsubstituted, branched or unbranched, cyclic or acyclicaliphatic, substituted or unsubstituted aromatic group, substituted orunsubstituted heteroaromatic group, or a substituted or unsubstitutedheterocyclic group. In one embodiment, R₁ is hydrogen, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, benzyl, or bromobenzyl;

R₂ is a substituted or unsubstituted, branched or unbranched, cyclic oracyclic aliphatic group, a substituted or unsubstituted aromatic group,a substituted or unsubstituted heteroaromatic group, or a substituted orunsubstituted heterocyclic group.

In one embodiment, R₂ is methyl, ethyl, 2-hydroxyethyl, isobutyl, afatty acid, a substituted or unsubstituted benzyl, a substituted orunsubstituted aryl, cysteine, homocysteine, or glutathione.

In one embodiment, the derivatives of romidepsin are of formulae (V) or(V′):

wherein:

each of R₁, R₂, R₃ and R₄ is the same or different and represent anamino acid side chain moiety;

each R₆ is the same or different and represents hydrogen or(C₁-C₄)alkyl; and

Pr¹ and Pr² are the same or different and represent hydrogen orthiol-protecting group.

In one embodiment, the amino acid side chain moieties are those derivedfrom natural amino acids. In one embodiment, the amino acid side chainmoieties are those derived from unnatural amino acids.

In one embodiment, each amino acid side chain is a moiety selected fromhydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, -L-O—C(0)-R′, -L-C(0)-0-R″,-L-A, -L-NR″R″, -L-Het-C(0)-Het-R″, and -L-Het-R″, wherein L is a(C₁-C₆)alkylene group, A is phenyl or a 5- or 6-membered heteroarylgroup, each R′ is the same or different and represents (C₁-C₄)alkyl,each R″ is the same or different and represent H or (C₁-C₆)alkyl, each-Het- is the same or different and is a heteroatom spacer selected from-0-, —N(R′″)—, and —S—, and each R′″ is the same of different andrepresents hydrogen or (C₁-C₄)alkyl.

In one embodiment, R₆ is hydrogen.

In one embodiment, Pr¹ and Pr² are the same or different and areselected from hydrogen and a protecting group selected from a benzylgroup which is optionally substituted by (C₁-C₆)alkoxy, (C₁-C₆)acyloxy,hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl,phenyl, t-butyl, adamanthyl, (C₁-C₆)acyloxymethyl, (C₁-C₆)alkoxymethyl,tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine,acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyland its derivatives, benzoyl and its derivatives, carbamoyl,phenylcarbamoyl, and (C₁-C₆)alkylcarbamoyl.

Various romidepsin derivatives of formula (V) and (V′) are disclosed inPCT application publication WO 2006/129105, published Dec. 7, 2006,which is incorporated herein by reference.

Romidepsin Formulation

In one embodiment, romidepsin is formulated for injection as a sterilelyophilized white powder and is supplied in a single-use vial containing10 mg romidepsin and 20 mg povidone, USP. The diluent is a sterile clearsolution and is supplied in a single-use vial containing a 2 mldeliverable volume. The diluent for romidepsin contains 80% (v/v)propylene glycol, USP and 20% (v/v) dehydrated alcohol, USP. Romidepsinis supplied as a kit containing two vials.

Romidepsin for injection is intended for intravenous infusion afterreconstitution with the supplied Diluent and after further dilution with0.9% Sodium Chloride, USP.

Methods of Use

It has been found that high levels of TSPYL5 expression in a cancerpatient's tumor show a high degree of correlation with resistance totreatment with an HDAC inhibitor This finding advantageously provides asignificant advancement in cancer management because it allows for theidentification of a patient population with increased likelihood ofpositive response to the treatment, by removal of patients with HDACinhibitor resistance.

A patient found to have low levels of TSPYL5 expression relative to anexpression threshold, is classified as being most likely to beresponsive to an HDAC inhibitor therapy, for example, romidepsintherapy. In one embodiment, provided herein are methods for selecting acancer patient that is a candidate for an HDAC inhibitor therapy basedon a gene expression signature of TSPYL5, comprising obtaining a tumorsample from said cancer patient, quantifying the level of TSPYL5expression in the tumor sample, wherein a low level of TSPYL5expression, relative to an expression threshold, correlates withincreased likelihood of sensitivity to the HDAC inhibitor therapy, andapplying said correlation to select the cancer patient that is acandidate for the HDAC inhibitor therapy. In one embodiment, the HDACinhibitor is romidepsin. In a further embodiment, the method encompassesthe additional step of initiating HDAC inhibitor therapy for said cancerpatient, for example, romidepsin therapy. In a further embodiment, themethod encompasses the additional step of administering atherapeutically effective amount of an HDAC inhibitor, for example,romidepsin, to said cancer patient.

A tumor found to have high levels of TSPYL5 expression relative to anexpression threshold, is classified as being most likely to be resistantto an HDAC inhibitor therapy, for example, romidepsin therapy. In oneembodiment, provided herein are methods for deselecting a cancer patientat risk for resistance to an HDAC inhibitor therapy as a candidate HDACinhibitor therapy based on a level of TSPYL5 expression, comprisingobtaining a tumor sample from said cancer patient, quantifying the levelof the TSPYL5 expression in the tumor sample, wherein a high level ofthe TSPYL5 expression, relative to an expression threshold, correlateswith resistance to the HDAC inhibitor therapy, and applying saidcorrelation to deselect the cancer patient at risk for resistance to theHDAC inhibitor therapy as a candidate HDAC inhibitor therapy. In oneembodiment, the HDAC inhibitor is romidepsin.

In one embodiment, provided herein are methods for confirmingtherapeutic efficacy of an HDAC inhibitor therapy in a cancer patientbased on a gene expression signature of the TSPYL5, comprising obtaininga tumor sample from the patient, detecting the presence of the TSPYL5expression in the tumor sample, quantifying the level of the TSPYL5expression in the tumor sample, wherein a low level of the TSPYL5expression, relative to an expression threshold, correlates withincreased likelihood of sensitivity to the HDAC inhibitor therapy, andapplying said correlation to confirm the therapeutic efficacy of theHDAC inhibitor therapy for said cancer patient. In one embodiment, theHDAC inhibitor is romidepsin. In a further embodiment, the methodencompasses the additional step of initiating HDAC inhibitor therapy forsaid cancer patient, for example, romidepsin therapy. In a furtherembodiment, the method encompasses the additional step of administeringa therapeutically effective amount of an HDAC inhibitor, for example,romidepsin, to said cancer patient.

In one embodiment, provided herein are methods for predicting a lack oftherapeutic efficacy of an HDAC inhibitor therapy in a cancer patientbased on a gene expression signature of the TSPYL5, comprising obtaininga tumor sample from the patient, quantifying the level of the TSPYL5expression in the tumor sample, wherein a high level of the TSPYL5expression, relative to an expression threshold, correlates withresistance to the HDAC inhibitor therapy, and applying said correlationto predict a lack of therapeutic efficacy of the HDAC inhibitor therapyfor said cancer patient. In one embodiment, the HDAC inhibitor isromidepsin.

Selection of a Patient Population

Classification of a particular patient population requires comparing thelevel of TSPYL5 expression in the tumor cells of a patient to anexpression threshold (also referred to as basal level). This expressionthreshold is a level of expression of TSPYL5 that can be used toevaluate whether the level of expression of TSPYL5 in tumor cells of apatient is low or high. Specifically, when the level of TSPYL5expression in the tumor cells of a patient is higher than the expressionthreshold, the cells are considered to have a high level of expression.Conversely, when the level of TSPYL5 expression in the tumor cells of apatient is lower than the expression threshold, the cells are consideredto have a low level of expression. Such high or low expression is nottypically calculated in terms of absolute TSPYL5 gene expression orprotein levels, but is determined using relative measurements. Theexpression threshold may be determined by a plurality of methods and isdetermined in tumor cells.

The expression threshold value provides a level of TSPYL5 expressionabove which exists a group of patients having a different resistance toHDAC inhibitor treatment than another group of patients having TSPYL5expression levels at or below the expression threshold. In oneembodiment, the expression threshold is a level of TSPYL5 expression ofin vitro cultured cells which may or may not have been manipulated tosimulate tumor cells.

Expression thresholds are not necessarily the levels of TSPYL5expression found in culture cell lines used to provide internalstandards. In one embodiment, these thresholds are determined based onlevels of TSPYL5 expression in tumor cells, for example, patient tumorsamples.

In one embodiment, the expression threshold is determined by comparisonof TSPYL5 expression levels in populations of patients having the sametype of cancer. In one embodiment, it is accomplished by histogramanalysis, in which the entire cohort of patients tested are graphicallypresented, wherein a first axis represents the levels of TSPYL5expression, and a second axis represents the number of patients in thecohort whose tumor cells express TSPYL5 at a given level. Two or moreseparate groups of patients are determined by identification of subsetspopulations of the cohort which have the same or similar expressionlevels of TSPYL5. Determination of the expression thresholds is madebased on an expression level which best distinguishes these separategroups.

Verification that the expression threshold distinguishes the likelihoodof responsiveness to HDAC inhibitor therapy in cancer patientsexpressing at or below-expression thresholds of TSPYL5 versus cancerpatients expressing above-expression thresholds of TSPYL5 is carried outusing single variable or multivariable analysis. These methods determinethe likelihood of a correlation between one or more variables and agiven outcome. In one embodiment, the methods determine the likelihoodof a correlation between TSPYL5 expression levels and resistance orresponsiveness to HDAC inhibitor therapy. Any one of a plurality ofmethods well known to those of ordinary skill in the art for carryingout these analyses may be used.

In one embodiment, population-based determination of expressionthresholds (i.e., histogram analysis) is carried out using a cohort ofpatients sufficient in size in order to determine two or more separategroups of patients having different TSPYL5 expression levels. In oneembodiment, such a cohort comprises at least 10 patients. In yet anotherembodiment, such a cohort comprises at least 27 patients. In anotherembodiment, such a cohort comprises at least 100 patients. In oneembodiment, verification of determined expression thresholds comprisesat least 10 patients. In another embodiment, it comprises at least 50patients. In yet another embodiment, it comprises at least 75 patients.In another embodiment, it is at least 100 patients.

In one embodiment, the expression threshold is a single value, equallyapplicable to every patient. In another embodiment, the expressionthreshold varies according to specific subpopulations of patients. Forexample, men might have a different expression threshold than women forthe same cancer type.

In one embodiment, the expression threshold of TSPYL5 expression is usedin conjunction with another variable found to be a statisticallysignificant indicator of the likelihood of resistance to HDAC inhibitortherapy. Such indicators include, but are not limited to, clinical orpathological indicators such as age, tumor size, tumor histology,clinical stage, and the like.

The TSPYL5 expression levels can be detected or quantitated by anymethods known in the art. In certain embodiments, antibody-based methodsare used. In certain embodiments, the detecting or quantitating methodis immunoblotting (western blot), an enzyme-linked immunosorbent assay(ELISA), immunohistochemistry, flow cytometry, a cytometric bead array,polymerase chain reaction or mass spectroscopy. In one embodiment,TSPYL5 expression levels in a tumor sample are measured usinganti-TSPYL5 antibodies.

In one embodiment, a patient is classified into a group having a certainlikelihood of resistance to HDAC inhibitor therapy based ondetermination of the level of TSPYL5 expression and comparison to anexpression threshold. The likelihood of resistance to HDAC inhibitortherapy for the patient is assessed based on likelihood of resistancefor patients in that group.

In one embodiment, a patient is classified into a group having a certainlikelihood of responsiveness to HDAC inhibitor therapy based ondetermination of level of TSPYL5 expression and comparison to anexpression threshold. The likelihood of responsiveness to HDAC inhibitortherapy for the patient is assessed based on likelihood ofresponsiveness for patients in that group.

In one embodiment, provided herein is a method for screening a cancerpatient to determine the risk of resistance to an HDAC inhibitortherapy. The method comprises determining the level of TSPYL5 expressionin a tumor sample or circulating tumor cell from the patient. A patientfound to have high levels of TSPYL5 expression relative to an expressionthreshold, is classified as being most likely resistant to HDACinhibitor therapy. In one embodiment, the HDAC inhibitor is romidepsin.In a further embodiment, the method encompasses the additional step ofadministering a therapeutically effective amount of a anticancer drugother than an HDAC inhibitor to said cancer patient.

In one embodiment, provided herein is a method for screening a cancerpatient to determine the likelihood of being responsive to an anticancertherapy with HDAC inhibitor. The method comprises determining the levelof TSPYL5 expression in a tumor sample or body fluid from the patient. Apatient found to have low levels of TSPYL5 expression relative to anexpression threshold, is classified as being most likely responsive toan HDAC inhibitor therapy. In one embodiment, the HDAC inhibitor isromidepsin. In a further embodiment, the method encompasses theadditional step of administering a therapeutically effective amount ofan HDAC inhibitor, for example, romidepsin, to said cancer patient.

Determination of Levels of TSPYL5 Expression

Determination of TSPYL5 expression is performed quantatively such thatthe level of expression can be determined. The TSPYL5 expression levelis used to predict resistance of a cancer patient to the HDAC inhibitortherapy based on the correlations provided herein. In one embodiment, ithas been found that when the TSPYL5 expression level is equal to orlower than an expression threshold of the TSPYL5 expression, a cancerpatient is more likely to be responsive to the HDAC inhibitor therapy,for example, romidepsin therapy, compared to the average likelihood ofresponsiveness to HDAC inhibitor therapy calculated from a random poolof cancer patients. In another embodiment, it has been found that whenthe TSPYL5 expression level is higher than an expression threshold ofthe TSPYL5 expression, a cancer patient is likely to be resistant to theHDAC inhibitor therapy, for example, romidepsin therapy.

Nucleotide and protein sequences for human TSPYL5 can be found, forexample, on the world wide web (ncbi.nlm.nih.gov) in the GenBankdatabase maintained by the National Center for Biotechnology (NCBI)under NCBI Reference Sequence: NM_(—)033512.2 and/or Gene-ID:85453.

In one embodiment, determination of TSPYL5 gene expression levels isperformed by one or more of the methods known to one skilled in the art.In one embodiment, expression of TSPYL5 is quantified at the proteinlevel. In one embodiment, the determination of the level of TSPYL5expression is based on the use of an antibody. In another embodiment,expression of TSPYL5 is quantified at the RNA level.

In one embodiment, levels of TSPYL5 protein expression are detected byusing antibodies, both monoclonal and polyclonal. In this embodiment,antibodies are used as specifically binding agents which bind TSPYL5protein or a polypeptide fragment thereof. Levels of TSPYL5 expressioncan be measured in a tumor sample using various art known methods. Forexample, quantitative PCR can be used to quantify TSPYL5 expressionlevels in circulating tumor cells in body fluids, such as blood.

In one embodiment, one or more of the TSPYL5 specific binding agents areused in a single assay to determine TSPYL5 protein levels. A certainprotein known to interact with a specific portion of the TSPYL5 proteinis coupled with another specifically binding protein. Using twoantibodies in a single assay, the specific levels of the differentlytranslated TSPYL5 polypeptides are measured by differentially measuringtwo antibodies. Preparation of the agent for use in detection of TSPYL5protein is carried out by the methods known to those skilled in the art(for example, the methods exemplified in the Current Protocols inMolecular Biology, John Wiley & Sons, 1999).

In another embodiment, detection of TSPYL5 protein levels is carried outby methods known to a skilled artisan, such as histochemical staining,Western Blot Analysis, or immunoprecipitation. In one embodiment, themethod of detecting TSPYL5 protein levels is an immunoassay, such asELISA, immuno-PCR, or the like.

In one embodiment, measuring levels of TSPYL5 mRNA includes detection ofhybridization or amplification with the mRNA. This detection is carriedout by analysis of mRNA either in vitro or in a tissue sample using oneof the methods known to those skilled in the art, such as quantitativePCR, gene chip arrays, etc. (Current Protocols in Molecular Biology,supra).

In one embodiment, provided herein is an array of probes for determiningthe level of TSPYL5 gene expression in a tumor sample by hybridizingwith one or more of the polynucleotides of TSPYL5 under stringent assayconditions; wherein the level of the TSPYL5 expression is used toidentify a cancer patient at risk for resistance to HDAC inhibitortherapy.

In another embodiment, provided herein is an array of probes fordetermining the level of TSPYL5 gene expression in a tumor sample byhybridizing with one or more mRNAs of the TSPYL5 under stringent assayconditions, wherein the level of the TSPYL5 expression is used toidentify a cancer patient at risk for resistance to HDAC inhibitortherapy.

In a further embodiment, provided herein is an array of antibodies fordetermining the level of TSPYL5 protein expression in a tumor sample,wherein the level of the TSPYL5 expression is used to identify a cancerpatient at risk for resistance to HDAC inhibitor therapy.

Methods of Treatment

In certain embodiments, provided are methods of treating cancer. Canceris a group of diseases which are characterized by uncontrolled growthand spread of abnormal cells. Cancers include, but are not limited to,carcinomas, sarcomas, leukemias, lymphomas and the like. In certainembodiments, cancer is a hematological malignancy. In certainembodiments, cancer is a solid tumor.

In certain embodiments the present disclosure relates to treatment ofhematological malignancies. Manifestations of hematological malignanciesinclude circulating malignant cells and malignant masses. Hematologicalmalignancies are types of cancers that affect the blood, bone marrow,and/or lymph nodes. Hematological malignancies that may be treated usingromidepsin include, but are not limited to: acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma(CTCL), peripheral T-cell lymphoma (PTCL), multiple myeloma, andmyelodysplastic syndromes. In certain embodiments, romidepsin is used totreat multiple myeloma. In certain particular embodiments, the cancer isrelapsed and/or refractory multiple myeloma. In other embodiments,romidepsin is used to treat chromic lymphocytic leukemia (CLL). Incertain particular embodiments, the cancer is relapsed and/or refractoryCLL. In other embodiments, romidepsin is used to treat chromicmyelogenous leukemia (CML). In certain embodiments, romidepsin is usedto treat acute lymphoblastic leukemia (ALL). In certain embodiments,romidepsin is used to treat acute myelogenous leukemia (AML). In certainembodiments, the cancer is cutaneous T-cell lymphoma (CTCL). In otherembodiments, the cancer is peripheral T-cell lymphoma (PTCL). In certainembodiments, the cancer is a myelodysplastic syndrome.

In some embodiments of the present disclosure, cancers treated include,but are not limited to, leukemias and lymphomas such as cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma, lymphomas associated withhuman T-cell lymphotropic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), B-cell lymphomas, acute lymphocytic leukemia,acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease,non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndromes.

In some such embodiments the disclosure relates to treatment of solidtumors such as lung, breast, colon, liver, pancreas, renal, prostate,ovarian, and/or brain. In some embodiments, the disclosure relates totreatment of pancreatic cancer. In some embodiments, the disclosurerelates to treatment of renal cancer. In some embodiments, thedisclosure relates to treatment of prostate cancer. In some embodiments,the disclosure relates to treatment of sarcomas. In some embodiments,the disclosure relates to treatment of soft tissue sarcomas.

In some embodiments, cancers that can be treated are solid cancers thatinclude, but are not limited to, mesothelioma, common solid tumors ofadults such as head and neck cancers (e.g., oral, laryngeal andesophageal), genitourinary cancers (e.g., prostate, bladder, renal,uterine, ovarian, testicular, rectal and colon), melanoma and other skincancers, stomach cancer, brain tumors, liver cancer and thyroid cancer,and/or childhood solid tumors such as brain tumors, neuroblastoma,retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas.).In certain embodiments, the cancer is melanoma. In other embodiments,the cancer is gastric cancer. In some embodiments, the disclosurerelates to treatment of solid tumors.

Cancers that may be treated using the methods provided herein, includingcombination therapy, include but not limited to, colon cancer, lungcancer, bone cancer, pancreatic cancer, stomach cancer, esophagealcancer, skin cancer, brain cancer, liver cancer, ovarian cancer,cervical cancer, uterine cancer, testicular cancer, prostate cancer,bladder cancer, kidney cancer, and neuroendocrine cancer.

In certain embodiments, cancer is pancreatic cancer. In certainembodiments, cancer is prostate cancer. In certain specific embodiments,the prostate cancer is hormone refractory prostate cancer.

In some particular embodiments, provided are methods to treat leukemias.In some embodiments, leukemia is chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute lymphocytic leukemia, acute myelogenousleukemia, or adult T cell leukemia/lymphoma.

In some embodiments, provided are methods of treating lymphomas. In someembodiments, lymphoma is Hodgkin's or non-Hodgkin's (e.g., T-celllymphomas such as peripheral T-cell lymphoma, cutaneous T-cell lymphoma,etc.) lymphoma.

In some embodiments, the disclosure relates to the treatment of multiplemyeloma and/or myelodysplastic syndromes.

An HDAC inhibitor may be administered using different routes ofadministration including, but not limited to, oral, rectal,transmucosal, transdermal, intestinal, and parenteral. In oneembodiment, the HDAC inhibitor is romidepsin.

In one embodiment, romidepsin is administered intravenously. In oneembodiment, romidepsin is administered intravenously over a time periodless than about 1 hour. In one embodiment, romidepsin is administeredintravenously over a 1-6 hour period. In one embodiment, romidepsin isadministered intravenously over a 3-4 hour period. In one embodiment,romidepsin is administered intravenously over a 5-6 hour period. In oneembodiment, romidepsin is administered intravenously over a 4 hourperiod.

In one embodiment, romidepsin is administered in a dose ranging from 0.5mg/m² to 28 mg/m². In one embodiment, romidepsin is administered in adose ranging from 0.5 mg/m² to 5 mg/m². In one embodiment, romidepsin isadministered in a dose ranging from 1 mg/m² to 25 mg/m². In oneembodiment, romidepsin is administered in a dose ranging from 1 mg/m² to20 mg/m². In one embodiment, romidepsin is administered in a doseranging from 1 mg/m² to 15 mg/m². In one embodiment, romidepsin isadministered in a dose ranging from 2 mg/m² to 15 mg/m². In oneembodiment, romidepsin is administered in a dose ranging from 2 mg/m² to12 mg/m². In one embodiment, romidepsin is administered in a doseranging from 4 mg/m² to 12 mg/m². In one embodiment, romidepsin isadministered in a dose ranging from 6 mg/m² to 12 mg/m². In oneembodiment, romidepsin is administered in a dose ranging from 8 mg/m² to12 mg/m². In one embodiment, romidepsin is administered in a doseranging from 8 mg/m² to 10 mg/m². In one embodiment, romidepsin isadministered in a dose of about 8 mg/m². In one embodiment, romidepsinis administered in a dose of about 9 mg/m². In one embodiment,romidepsin is administered in a dose of about 10 mg/m². In oneembodiment, romidepsin is administered in a dose of about 11 mg/m². Inone embodiment, romidepsin is administered in a dose of about 12 mg/m².In one embodiment, romidepsin is administered in a dose of about 13mg/m². In one embodiment, romidepsin is administered in a dose of about14 mg/m². In one embodiment, romidepsin is administered in a dose ofabout 15 mg/m².

In one embodiment, romidepsin is administered in a dose of 14 mg/m² asan IV infusion over a 4 hour period on days 1, 8 and 15 of the 28 daycycle. In one embodiment, the cycle is repeated every 28 days.

In one embodiment, increasing doses of romidepsin are administered overthe course of a cycle. In one embodiment, the dose of about 8 mg/m²followed by a dose of about 10 mg/m², followed by a dose of about 12mg/m² is administered over a cycle.

In one embodiment, romidepsin is administered orally. In one embodiment,romidepsin is administered orally on a daily basis. In certainembodiments, romidepsin is dosed orally in the range of 10 mg/m² to 300mg/m². In certain embodiments, romidepsin is dosed orally in the rangeof 25 mg/m² to 100 mg/m². In certain embodiments, romidepsin is dosedorally in the range of 100 mg/m² to 200 mg/m². In certain embodiments,romidepsin is dosed orally in the range of 200 mg/m² to 300 mg/m². Incertain embodiments, romidepsin is dosed orally at greater than 300mg/m². In certain embodiments, romidepsin is dosed orally in the rangeof 50 mg/m² to 150 mg/m². In other embodiments, the oral dosage rangesfrom 25 mg/m² to 75 mg/m². In one embodiment, romidepsin is administeredorally every other day. In one embodiment, romidepsin is administeredorally every third, fourth, fifth, or sixth day. In one embodiment,romidepsin is administered orally every week. In one embodiment,romidepsin is administered orally every other week.

In one embodiment, romidepsin is administered orally in a dose of 50mg/m² on days 1, 8 and 15 of the 28 day cycle. In one embodiment, thecycle is repeated every 28 days.

In one embodiment, increasing doses of romidepsin are administered overthe course of a cycle. In one embodiment, the dose of about 25 mg/m²followed by a dose of about 50 mg/m², followed by a dose of about 75mg/m² is administered over a cycle.

In one embodiment, one cycle comprises the administration of from about25 to about 150 mg/m² of romidepsin daily for three to four weeks andthen one or two weeks of rest. In one embodiment, the number of cyclesduring which the treatment is administered to a patient will be fromabout one to about 40 cycles, or from about one to about 24 cycles, orfrom about two to about 16 cycles, or from about four to about threecycles.

Dosing

In some embodiments, romidepsin and/or compositions comprisingromidepsin are administered according to a standard dosing regimen. Insome embodiments, romidepsin and/or compositions comprising romidepsinare administered according to an accelerated dosing regimen.

Standard Dosing for Romidepsin

In some embodiments, unit doses of romidepsin are within the range ofabout 0.5 mg/m² to about 28 mg/m² body surface area. In someembodiments, the range of about 6 mg/m² to about 18 mg/m² is used. Insome embodiments, the range is about 10 mg/m² to about 17 mg/m². In someembodiments, particular unit doses are 10 mg/m², 12 mg/m², 13 mg/m², 14mg/m², and 15 mg/m².

In some embodiments, intravenous dosing regimens include daily dosingfor 2 weeks, twice weekly dosing for 4 weeks, thrice weekly dosing for 4weeks, and various other intermittent schedules (e.g., on days 1, 3, and5; on days 4 and 10; on days 1, 8 and 15; on days 1 and 15; on days 5and 12; or on days 5, 12, and 19 of 21 or 28 day cycles).

In some embodiments, romidepsin is administered in individual unit dosesover 4 hours on days 1, 8, and 15, with courses repeating every 28 days.Often, several courses (e.g., at least 4, at least 6, or more) areadministered. Indeed, instances have been reported of as many as 72courses being administered. In some embodiments, individual unit dosesare administered by 4 hour infusion.

Accelerated Dosing for Romidepsin

Accelerated dosing regimens for romidepsin may be utilized, in which oneor more individual unit doses is administered intravenously over aperiod of time that is less than or equal to about one hour. In someembodiments, one or more individual doses are administered intravenouslyover a period of time that is less than about 50 minutes, 40 minutes, 30minutes, 20 minutes, or less. Any regimen that includes at least oneunit dose administered over a period of time that is less than about onehour (60 minutes) may be considered an accelerated dosing regimen inaccordance with the present disclosure.

In some embodiments, all unit doses within a regimen are administeredintravenously over a time period that is less than or equal to about onehour. In some embodiments, only some of the unit doses within a regimenare administered over a time period that is less than or equal to aboutone hour. In some embodiments, one or more unit doses within a regimenare administered by a route other than intravenous administration (e.g.,oral, subcutaneous, nasal, topical, etc.).

Accelerated dosing regimens of romidepsin can be administered without asignificant increase in toxicity or adverse events, particularly inserious adverse events, as compared with a comparable regimen (e.g., anotherwise identical regimen) in which individual unit doses areadministered intravenously over a 4-hour period. In one embodiment,accelerated dosing regimens can be administered without a significantincrease in toxicity or adverse events, particularly in serious adverseevents, as compared with a standard regimen of romidepsin administeredby 4-hour intravenous infusion of a dose of about 6-14 mg/m² on days 1,8, and 15 of a 28 day cycle.

In some embodiments, romidepsin is administered in an accelerated dosingregimen that is identical to a standard dosing regimen except that oneor more unit doses is administered over a time period that is less thanabout 1 hour (e.g., rather than over a time period of about 4 hours).

As will be appreciated by one of skill in the art, the dosage, timingand/or routes of administration of particular unit doses of romidepsinmay vary depending on the patient and condition being treated. Incertain embodiments, the cycles are continued as long as the patient isresponding. Therapy may be terminated once there is disease progression,a cure or remission is achieved, or side effects become intolerable.Adverse side effects may also call for lowering the dosage of romidepsinadministered, or for adjusting the schedule by which doses areadministered.

Pharmaceutical Formulations

In one embodiment, provided herein are pharmaceutical formulations,which comprise romidepsin, or a pharmaceutically acceptable salt orsolvate thereof, as an active ingredient, in combination with one ormore pharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition comprises at least one nonrelease controllingexcipient or carrier. In one embodiment, the pharmaceutical compositioncomprises at least one release controlling and at least one nonreleasecontrolling excipients or carriers.

In certain embodiments, romidepsin used in the pharmaceuticalcompositions provided herein is in a solid form. Suitable solid formsinclude, but are not limited to, solid forms comprising romidepsin, andsolid forms comprising salts of romidepsin. In certain embodiments,solid forms provided herein include polymorphs, solvates (includinghydrates), and cocrystals comprising romidepsin and/or salts thereof. Incertain embodiments, the solid form is an amorphous form of romidepsin,or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the pharmaceutical compositions provided herein maybe formulated in various dosage forms for parenteral administration. Inone embodiment, the pharmaceutical compositions provided herein may beprovided in a unit-dosage form or multiple-dosage form. A unit-dosageform, as used herein, refers to a physically discrete unit suitable foradministration to human and animal subjects, and packaged individuallyas is known in the art. Each unit-dose contains a predetermined quantityof the active ingredient(s) sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarriers or excipients. Examples of a unit-dosage form include anampoule, syringe, and individually packaged tablet and capsule. Aunit-dosage form may be administered in fractions or multiples thereof.A multiple-dosage form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dosage form. Examples of a multiple-dosage form include a vial,bottle of tablets or capsules, or bottle of pints or gallons.

In one embodiment, the pharmaceutical compositions provided herein maybe administered at once or multiple times at intervals of time. It isunderstood that the precise dosage and duration of treatment may varywith the age, weight, and condition of the patient being treated, andmay be determined empirically using known testing protocols or byextrapolation from in vivo or in vitro test or diagnostic data. It isfurther understood that for any particular individual, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the formulations.

In some embodiments, the composition is prepared by lyophilization froma solution. In particular embodiments, the composition is prepared bylyophilization from a solution of t-butanol and water. In someembodiments, the solvent is tert-butanol. In some embodiments, thesolvent is a mixture of tert-butanol and water. In some embodiments, thesolution is (60:40) (v/v) of t-butanol and water. In some embodiments,the pH adjustor is inorganic acid. In one embodiment, the inorganic acidis hydrochloric acid.

Parenteral Administration

In one embodiment, the pharmaceutical compositions provided herein maybe administered parenterally by injection, infusion, or implantation,for local or systemic administration. Parenteral administration, as usedherein, include intravenous, intraarterial, intraperitoneal,intrathecal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intrasynovial, and subcutaneousadministration.

In one embodiment, the pharmaceutical compositions provided herein maybe formulated in any dosage forms that are suitable for parenteraladministration, including solutions, suspensions, emulsions, micelles,liposomes, microspheres, nanosystems, and solid forms suitable forsolutions or suspensions in liquid prior to injection. Such dosage formscan be prepared according to conventional methods known to those skilledin the art of pharmaceutical science (see, e.g., Remington, The Scienceand Practice of Pharmacy, supra).

In one embodiment, the pharmaceutical compositions intended forparenteral administration may include one or more pharmaceuticallyacceptable carriers and excipients, including, but not limited to,aqueous vehicles, water-miscible vehicles, non-aqueous vehicles,antimicrobial agents or preservatives against the growth ofmicroorganisms, stabilizers, solubility enhancers, isotonic agents,buffering agents, antioxidants, local anesthetics, suspending anddispersing agents, wetting or emulsifying agents, complexing agents,sequestering or chelating agents, cryoprotectants, lyoprotectants,thickening agents, pH adjusting agents, and inert gases.

In one embodiment, suitable aqueous vehicles include, but are notlimited to, water, saline, physiological saline or phosphate bufferedsaline (PBS), sodium chloride injection, Ringers injection, isotonicdextrose injection, sterile water injection, dextrose and lactatedRingers injection. Non-aqueous vehicles include, but are not limited to,fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil,olive oil, peanut oil, peppermint oil, safflower oil, sesame oil,soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, andmedium-chain triglycerides of coconut oil, and palm seed oil.Water-miscible vehicles include, but are not limited to, ethanol,1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol300 and polyethylene glycol 400), propylene glycol, glycerin,N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

In one embodiment, suitable antimicrobial agents or preservativesinclude, but are not limited to, phenols, cresols, mercurials, benzylalcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates,thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl-and propyl-parabens, and sorbic acid. Suitable isotonic agents include,but are not limited to, sodium chloride, glycerin, and dextrose.Suitable buffering agents include, but are not limited to, phosphate andcitrate. Suitable antioxidants are those as described herein, includingbisulfite and sodium metabisulfite. Suitable local anesthetics include,but are not limited to, procaine hydrochloride. Suitable suspending anddispersing agents are those as described herein, including sodiumcarboxymethylcelluose, hydroxypropyl methylcellulose, andpolyvinylpyrrolidone. Suitable emulsifying agents include thosedescribed herein, including polyoxyethylene sorbitan monolaurate,polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate.Suitable sequestering or chelating agents include, but are not limitedto EDTA. Suitable pH adjusting agents include, but are not limited to,sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.Suitable complexing agents include, but are not limited to,cyclodextrins, including α-cyclodextrin, β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, andsulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

In one embodiment, the pharmaceutical compositions provided herein maybe formulated for single or multiple dosage administration. The singledosage formulations are packaged in an ampoule, a vial, or a syringe.The multiple dosage parenteral formulations may contain an antimicrobialagent at bacteriostatic or fungistatic concentrations. All parenteralformulations must be sterile, as known and practiced in the art.

In one embodiment, the pharmaceutical compositions are provided asready-to-use sterile solutions. In another embodiment, thepharmaceutical compositions are provided as sterile dry solubleproducts, including lyophilized powders and hypodermic tablets, to bereconstituted with a vehicle prior to use. In yet another embodiment,the pharmaceutical compositions are provided as ready-to-use sterilesuspensions. In yet another embodiment, the pharmaceutical compositionsare provided as sterile dry insoluble products to be reconstituted witha vehicle prior to use. In still another embodiment, the pharmaceuticalcompositions are provided as ready-to-use sterile emulsions.

In one embodiment, the pharmaceutical compositions provided herein maybe formulated as immediate or modified release dosage forms, includingdelayed-, sustained, pulsed-, controlled, targeted-, andprogrammed-release forms.

In one embodiment, the pharmaceutical compositions may be formulated asa suspension, solid, semi-solid, or thixotropic liquid, foradministration as an implanted depot. In one embodiment, thepharmaceutical compositions provided herein are dispersed in a solidinner matrix, which is surrounded by an outer polymeric membrane that isinsoluble in body fluids but allows the active ingredient in thepharmaceutical compositions diffuse through.

In one embodiment, suitable inner matrixes includepolymethylmethacrylate, polybutyl-methacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethylene terephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers, such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinyl alcohol, andcross-linked partially hydrolyzed polyvinyl acetate.

In one embodiment, suitable outer polymeric membranes includepolyethylene, polypropylene, ethylene/propylene copolymers,ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers,silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinatedpolyethylene, polyvinylchloride, vinyl chloride copolymers with vinylacetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyloxyethanol copolymer,and ethylene/vinyl acetate/vinyl alcohol terpolymer.

Oral Administration

The pharmaceutical compositions provided herein for oral administrationcan be provided in solid, semisolid, or liquid dosage forms for oraladministration. As used herein, oral administration also includesbuccal, lingual, and sublingual administration. Suitable oral dosageforms include, but are not limited to, tablets, fastmelts, chewabletablets, capsules, pills, strips, troches, lozenges, pastilles, cachets,pellets, medicated chewing gum, bulk powders, effervescent ornon-effervescent powders or granules, oral mists, solutions, emulsions,suspensions, wafers, sprinkles, elixirs, and syrups. In addition to theactive ingredient(s), the pharmaceutical compositions can contain one ormore pharmaceutically acceptable carriers or excipients, including, butnot limited to, binders, fillers, diluents, disintegrants, wettingagents, lubricants, glidants, coloring agents, dye-migration inhibitors,sweetening agents, flavoring agents, emulsifying agents, suspending anddispersing agents, preservatives, solvents, non-aqueous liquids, organicacids, and sources of carbon dioxide.

Binders or granulators impart cohesiveness to a tablet to ensure thetablet remaining intact after compression. Suitable binders orgranulators include, but are not limited to, starches, such as cornstarch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500);gelatin; sugars, such as sucrose, glucose, dextrose, molasses, andlactose; natural and synthetic gums, such as acacia, alginic acid,alginates, extract of Irish moss, panwar gum, ghatti gum, mucilage ofisabgol husks, carboxymethylcellulose, methylcellulose,polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powderedtragacanth, and guar gum; celluloses, such as ethyl cellulose, celluloseacetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose, methyl cellulose, hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC);microcrystalline celluloses, such as AVICEL-PH-101, AVICEL-PH-103,AVICEL RC-581, AVICEL-PH-105 (FMC Corp., Marcus Hook, Pa.); and mixturesthereof. Suitable fillers include, but are not limited to, talc, calciumcarbonate, microcrystalline cellulose, powdered cellulose, dextrates,kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinizedstarch, and mixtures thereof. The amount of a binder or filler in thepharmaceutical compositions provided herein varies upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. The binder or filler may be present from about 50 to about 99%by weight in the pharmaceutical compositions provided herein.

Suitable diluents include, but are not limited to, dicalcium phosphate,calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose,kaolin, mannitol, sodium chloride, dry starch, and powdered sugar.Certain diluents, such as mannitol, lactose, sorbitol, sucrose, andinositol, when present in sufficient quantity, can impart properties tosome compressed tablets that permit disintegration in the mouth bychewing. Such compressed tablets can be used as chewable tablets. Theamount of a diluent in the pharmaceutical compositions provided hereinvaries upon the type of formulation, and is readily discernible to thoseof ordinary skill in the art.

Suitable disintegrants include, but are not limited to, agar; bentonite;celluloses, such as methylcellulose and carboxymethylcellulose; woodproducts; natural sponge; cation-exchange resins; alginic acid; gums,such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses,such as croscarmellose; cross-linked polymers, such as crospovidone;cross-linked starches; calcium carbonate; microcrystalline cellulose,such as sodium starch glycolate; polacrilin potassium; starches, such ascorn starch, potato starch, tapioca starch, and pre-gelatinized starch;clays; aligns; and mixtures thereof. The amount of a disintegrant in thepharmaceutical compositions provided herein varies upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. The amount of a disintegrant in the pharmaceutical compositionsprovided herein varies upon the type of formulation, and is readilydiscernible to those of ordinary skill in the art. The pharmaceuticalcompositions provided herein may contain from about 0.5 to about 15% orfrom about 1 to about 5% by weight of a disintegrant.

Suitable lubricants include, but are not limited to, calcium stearate;magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol;mannitol; glycols, such as glycerol behenate and polyethylene glycol(PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetableoil, including peanut oil, cottonseed oil, sunflower oil, sesame oil,olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyllaureate; agar; starch; lycopodium; silica or silica gels, such asAEROSIL 200 (W.R. Grace Co., Baltimore, Md.) and CAB-O-SIL® (Cabot Co.of Boston, Mass.); and mixtures thereof. The pharmaceutical compositionsprovided herein may contain about 0.1 to about 5% by weight of alubricant.

Suitable glidants include, but are not limited to, colloidal silicondioxide, CAB-O-SIL® (Cabot Co. of Boston, Mass.), and asbestos-freetalc. Suitable coloring agents include, but are not limited to, any ofthe approved, certified, water soluble FD&C dyes, and water insolubleFD&C dyes suspended on alumina hydrate, and color lakes and mixturesthereof. A color lake is the combination by adsorption of awater-soluble dye to a hydrous oxide of a heavy metal, resulting in aninsoluble form of the dye. Suitable flavoring agents include, but arenot limited to, natural flavors extracted from plants, such as fruits,and synthetic blends of compounds which produce a pleasant tastesensation, such as peppermint and methyl salicylate. Suitable sweeteningagents include, but are not limited to, sucrose, lactose, mannitol,syrups, glycerin, and artificial sweeteners, such as saccharin andaspartame. Suitable emulsifying agents include, but are not limited to,gelatin, acacia, tragacanth, bentonite, and surfactants, such aspolyoxyethylene sorbitan monooleate (TWEEN® 20), polyoxyethylenesorbitan monooleate 80 (TWEEN® 80), and triethanolamine oleate. Suitablesuspending and dispersing agents include, but are not limited to, sodiumcarboxymethylcellulose, pectin, tragacanth, Veegum, acacia, sodiumcarbomethylcellulose, hydroxypropyl methylcellulose, andpolyvinylpyrrolidone.

Suitable preservatives include, but are not limited to, glycerin, methyland propylparaben, benzoic add, sodium benzoate and alcohol. Suitablewetting agents include, but are not limited to, propylene glycolmonostearate, sorbitan monooleate, diethylene glycol monolaurate, andpolyoxyethylene lauryl ether. Suitable solvents include, but are notlimited to, glycerin, sorbitol, ethyl alcohol, and syrup. Suitablenon-aqueous liquids utilized in emulsions include, but are not limitedto, mineral oil and cottonseed oil. Suitable organic acids include, butare not limited to, citric and tartaric acid. Suitable sources of carbondioxide include, but are not limited to, sodium bicarbonate and sodiumcarbonate.

It should be understood that many carriers and excipients may serve aplurality of functions, even within the same formulation.

The pharmaceutical compositions provided herein for oral administrationcan be provided as compressed tablets, tablet triturates, chewablelozenges, rapidly dissolving tablets, multiple compressed tablets, orenteric-coating tablets, sugar-coated, or film-coated tablets.Enteric-coated tablets are compressed tablets coated with substancesthat resist the action of stomach acid but dissolve or disintegrate inthe intestine, thus protecting the active ingredients from the acidicenvironment of the stomach. Enteric-coatings include, but are notlimited to, fatty acids, fats, phenyl salicylate, waxes, shellac,ammoniated shellac, and cellulose acetate phthalates. Sugar-coatedtablets are compressed tablets surrounded by a sugar coating, which maybe beneficial in covering up objectionable tastes or odors and inprotecting the tablets from oxidation. Film-coated tablets arecompressed tablets that are covered with a thin layer or film of awater-soluble material. Film coatings include, but are not limited to,hydroxyethylcellulose, sodium carboxymethylcellulose, polyethyleneglycol 4000, and cellulose acetate phthalate. Film coating imparts thesame general characteristics as sugar coating. Multiple compressedtablets are compressed tablets made by more than one compression cycle,including layered tablets, and press-coated or dry-coated tablets.

The tablet dosage forms can be prepared from the active ingredient inpowdered, crystalline, or granular forms, alone or in combination withone or more carriers or excipients described herein, including binders,disintegrants, controlled-release polymers, lubricants, diluents, and/orcolorants. Flavoring and sweetening agents are especially useful in theformation of chewable tablets and lozenges.

The pharmaceutical compositions provided herein for oral administrationcan be provided as soft or hard capsules, which can be made fromgelatin, methylcellulose, starch, or calcium alginate. The hard gelatincapsule, also known as the dry-filled capsule (DFC), consists of twosections, one slipping over the other, thus completely enclosing theactive ingredient. The soft elastic capsule (SEC) is a soft, globularshell, such as a gelatin shell, which is plasticized by the addition ofglycerin, sorbitol, or a similar polyol. The soft gelatin shells maycontain a preservative to prevent the growth of microorganisms. Suitablepreservatives are those as described herein, including methyl- andpropyl-parabens, and sorbic acid. The liquid, semisolid, and soliddosage forms provided herein may be encapsulated in a capsule. Suitableliquid and semisolid dosage forms include solutions and suspensions inpropylene carbonate, vegetable oils, or triglycerides. Capsulescontaining such solutions can be prepared as described in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient.

The pharmaceutical compositions provided herein for oral administrationcan be provided in liquid and semisolid dosage forms, includingemulsions, solutions, suspensions, elixirs, and syrups. An emulsion is atwo-phase system, in which one liquid is dispersed in the form of smallglobules throughout another liquid, which can be oil-in-water orwater-in-oil. Emulsions may include a pharmaceutically acceptablenon-aqueous liquid or solvent, emulsifying agent, and preservative.Suspensions may include a pharmaceutically acceptable suspending agentand preservative. Aqueous alcoholic solutions may include apharmaceutically acceptable acetal, such as a di(lower alkyl) acetal ofa lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and awater-miscible solvent having one or more hydroxyl groups, such aspropylene glycol and ethanol. Elixirs are clear, sweetened, andhydroalcoholic solutions. Syrups are concentrated aqueous solutions of asugar, for example, sucrose, and may also contain a preservative. For aliquid dosage form, for example, a solution in a polyethylene glycol maybe diluted with a sufficient quantity of a pharmaceutically acceptableliquid carrier, e.g., water, to be measured conveniently foradministration.

Other useful liquid and semisolid dosage forms include, but are notlimited to, those containing the active ingredient(s) provided herein,and a dialkylated mono- or poly-alkylene glycol, including,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 referto the approximate average molecular weight of the polyethylene glycol.These formulations can further comprise one or more antioxidants, suchas butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA),propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine,lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoricacid, bisulfite, sodium metabisulfite, thiodipropionic acid and itsesters, and dithiocarbamates.

The pharmaceutical compositions provided herein for oral administrationcan be also provided in the forms of liposomes, micelles, microspheres,or nanosystems. Micellar dosage forms can be prepared as described inU.S. Pat. No. 6,350,458.

The pharmaceutical compositions provided herein for oral administrationcan be provided as non-effervescent or effervescent, granules andpowders, to be reconstituted into a liquid dosage form. Pharmaceuticallyacceptable carriers and excipients used in the non-effervescent granulesor powders may include diluents, sweeteners, and wetting agents.Pharmaceutically acceptable carriers and excipients used in theeffervescent granules or powders may include organic acids and a sourceof carbon dioxide.

Coloring and flavoring agents can be used in all of the above dosageforms.

The pharmaceutical compositions provided herein for oral administrationcan be formulated as immediate or modified release dosage forms,including delayed-, sustained, pulsed-, controlled, targeted-, andprogrammed-release forms.

Kits

In one embodiment, a kit comprises a container filled with an HDACinhibitor, reagents for detecting the TSPYL5 gene and/or protein, andinstructions for detecting the level of expression of the TSPYL5 geneand/or protein in a patient having cancer.

In certain embodiments, the kit comprises one or more probes that bindspecifically to the TSPYL5 mRNAs. In certain embodiments, the kitfurther comprises a washing solution. In certain embodiments, the kitfurther comprises reagents for performing a hybridization assay, TSPYL5mRNA isolation or purification means, detection means, as well aspositive and negative controls. In certain embodiments, the kit furthercomprises an instruction for using the kit. The kit can be tailored forin-home use, clinical use, or research use.

In certain embodiments, provided herein is a kit for detecting theTSPYL5 protein level. In certain embodiments, the kits comprises adipstick coated with an antibody that recognizes the protein biomarker,washing solutions, reagents for performing the assay, protein isolationor purification means, detection means, as well as positive and negativecontrols. In certain embodiments, the kit further comprises aninstruction for using the kit. The kit can be tailored for in-home use,clinical use, or research use.

Such a kit can employ, for example, a dipstick, a membrane, a chip, adisk, a test strip, a filter, a microsphere, a slide, a multiwell plate,or an optical fiber. The solid support of the kit can be, for example, aplastic, silicon, a metal, a resin, glass, a membrane, a particle, aprecipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, acapillary, a film, a plate, or a slide. The biological sample can be,for example, a cell culture, a cell line, a tissue, an oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a urine sample, or a skin sample.

In one embodiment, a kit comprises a dosage form of romidepsin. Kits canfurther comprise a pharmacologically active derivative of romidepsin.

In other embodiments, kits can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, and drip bags.

In one embodiment, kits can further comprise a pharmaceuticallyacceptable vehicle that can be used to administer one or more activeingredients. For example, if an active ingredient is provided in a solidform that must be reconstituted for parenteral administration, the kitcan comprise a sealed container of a suitable vehicle in which theactive ingredient can be dissolved to form a particulate-free sterilesolution that is suitable for parenteral administration. Examples ofpharmaceutically acceptable vehicles include, but are not limited to:Water for Injection USP; aqueous vehicles such as, but not limited to,Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Unless otherwise defined, all technical and scientific terms used hereinare accorded the meaning commonly known to one of skill in the art. Allpublications, patents, published patent applications, and otherreferences mentioned herein are hereby incorporated by reference intheir entirety. The embodiments of the disclosure should not be deemedto be mutually exclusive and can be combined.

Examples

The following examples are provided by way of illustration, notlimitation.

Example 1 Definition and Calculation of S-Value

Sensitivity of cells to HDAC inhibitor treatment is expressed as theirS-value (S), which is a dimensionless compound value generated by addingthe values of IC₅₀ and the growth rate (GR) at the maximum drugconcentration used to determine the IC₅₀. The term (maximum drugconcentration/200) is added in the equation below to achieve equalweighting of the S-value for IC₅₀ and GR.

S=IC ₅₀+(GR+100)×(maximum drug concentration/200)

See FIG. 1 for an example of an S value calculation and explanation ofGR.

The S-Value is highly correlated with the Area Under the Curve (AUC)value obtained from inhibition vs. compound concentration graph, andthese two values can be used interchangeably.

To determine cell growth IC₅₀ and GR at a given drug concentration,growth was plotted against drug concentration as a percentage of thenumber of cells present at the beginning of the experiment. Cells wereplated in 96-well plates and allowed to attach and grow overnight. Thefollowing day, the cells were treated with serial dilutions of testcompound. The viable cells were measured at day 0 (24 hours afterplating) and day 3 (72 hours after adding the tested compound) usingCell Titer-Glo. Growth was calculated compared to day 0. Explanationsample graph is presented in FIG. 1.

The plot generated two key data, the IC₅₀ value and the % growth at thehighest tested compound concentration (GR). The highest concentrationtested for romidepsin was 1000 nM.

The IC₅₀s in these experiments could range from 0-1000 nM. A given IC₅₀value, 97.35 nM as shown in the sample plot (FIG. 1), was added as itsnumerical value in the calculation of the S-value, 97.35 (FIG. 1).Growth at the highest drug concentration tested ranged from −100 to+100. For the calculation of the S-Value, 100 was added to the numericalvalue of the observed growth, so that only positive numbers weregenerated (after adding 100, the range was from 0-200). Since IC₅₀s andgrowth at the maximum drug concentration tested have different numericalranges (0-1000 as compared to 0-200), the value for growth inhibitionwas multiplied by the maximum drug concentration used/200. This meansthat in the example given, both IC₅₀ and GR could contribute values of0-1000 to the S-value.

See FIG. 1:

$\begin{matrix}{{S\text{-}{Value}} = {{IC}_{50} + {( {{{Growth}\mspace{14mu} {inhibition}\mspace{14mu} {at}\mspace{14mu} 1000\mspace{14mu} {nM}} + 100} ) \times 5}}} \\{= {{97.35 + {( {{- 79.5} + 100} ) \times 5}} = {199.85.\quad}}}\end{matrix}\quad$

Example 2 Sensitivity to Romidepsin in Various Cell Lines

A cell line panel viability screen was used to compare the effects of a6 hour and 72 hour treatment with romidepsin across a wide variety ofcell lines. Although the majority of the tested cell lines showedsensitivity to romidepsin in the 72 hour assay, the 6 hour exposurerevealed significant differences in sensitivity to romidepsin in variouscell lines. The S-Value was computed for the 6 h treatment and rangedfrom 5 for the most sensitive cell lines to 2000 for the most resistantcell lines. The results are shown in FIGS. 2A and 2B. To identify geneswhose expression correlated with sensitivity to HDAC inhibitortreatment, we applied two way ANOVA to select genes that were correlatedwith sensitivity. We used 24 solid tumor cell lines of breast, colon andovary origin, since lines from these 3 tumor types had a large spread ofS values, and identified 254 unique probes that were significantlydifferent between these two groups. The most overexpressed gene in theresistant subgroup, ABCB1 (Res/Sen=29.6) is known to cause resistance toromidepsin treatment. This approach identified novel genes thatcorrelated with resistance and/or sensitivity to the HDAC inhibitors butnot tissue origin. Using this approach, it was found that the expressionof the TSPYL5 gene correlated with resistance to romidepsin.

Example 3 TSPYL5 Expression Correlates with Resistance to Romidepsin inCell Line Screen

A cell line panel viability screen was used to compare the effects ofromidepsin across a wide variety of cell lines and is shown in FIG. 3.

The data indicates that the expression of TSPYL5 correlated withresistance to romidepsin in the tested cell lines. It was demonstratedthat no cell line expressing high levels of TSPYL5 was sensitive totreatment with romidepsin or panobinostat, while all cell linessensitive to treatment with these two HDAC inhibitors had baselineexpression of TSPYL5.

Example 4 TSPYL5 Expression Correlates with Resistance to Romidepsin inPrimary Patient Tumor Explants

Primary human tumors were propagated in mice. Tumors were dissociatedand their sensitivity to romidepsin was tested using a clonogenic assay.

The clonogenic assay was performed in a 24-well format according to amodified two-layer soft agar assay introduced by Hamburger & Salmon(Hamburger A W, Salmon S E. Primary bioassay of human tumor stem cells.Science 197:461 (1977)). Briefly, cells were seeded on a bottom layer ofgrowth medium supplemented with 0.4% (w/v) agar. Cells were added in 0.2ml of the same medium supplemented with 0.4% (w/v) agar. Test compoundswere applied diluted in culture medium. Cultures were incubated at 37°C. and 7.5% CO₂ in a humidified atmosphere for up to 21 days. After 4-21days, colonies were counted and drug effect was expressed as percentcolony formations compared to untreated controls.

The graph shown in FIG. 4 shows the expression of TSPYL5 against theromidepsin IC₅₀ obtained with the clonogenic assay. Primary tumors withhigh TSPYL5 expression were resistant to treatment with romidepsin.

Example 5 Effect of Knockdown of TSPYL5 on Sensitivity to Romidepsin

It was shown that knockdown of TSPYL5 expression increased sensitivityto romidepsin treatment. Results are shown in Table 1 below and in FIG.5.

TABLE 1 Knockdown of TSPYL5 in SKOV3 cells with shRNA increasessensitivity to romidepsin Cell line IC50, nM GR, % S value SKOV3 TSPYL5KD 58 −88.6 115 71 −93.2 105 43 −86.1 112 57 −84.3 135 118 −92.5 156SKOV3 NS 141 −70 291 136 −76.5 253 139 −76.6 256 98 −73.5 230 162 −85.1243

Effect of knocking down TSPYL5 expression using shRNA on IC₅₀, GR and Svalues of romidepsin treatment. SKOV3 cells expressing either TSPYL5shRNA (SKOV3 TSPYL5 KD) or non-silencing control shRNA (SKOV3 NS) werecreated using lentiviral infection. Knockdown of TSPYL5 in SKOV3 TSPYL5KD cells was verified using western blot and quantitative PCR.Expression of TSPYL5 in these cells was reduced by 70%. After selectionof stable pools with puromycin, cells were treated with varyingconcentrations of romidepsin for determination of IC₅₀, GR and S value.Data from 5 independent experiments are shown. SKOV3 TSPYL5 KD cellshave lower IC₅₀s, show stronger growth rate (GR) and have thus smaller Svalues than SKOV3 NS cells, meaning these cells are more sensitive totreatment with romidepsin. The data are plotted in FIG. 5. Differencesbetween the 2 lines are significant (paired t test).

Example 6 Effect of Knockdown of TSPYL5 on Sensitivity to Panobinostat

It was shown that knockdown of TSPYL5 with shRNA increases sensitivityof SKOV3 cells to panobinostat treatment. The results are shown in FIG.7.

Example 7 Effect of Knockdown of TSPYL5 on Sensitivity of Human DermalFibroblasts to Romidepsin

It was shown that knockdown of TSPYL5 with shRNA increases sensitivityof human dermal fibroblast (HDF) cells to romidepsin treatment. Theresults are shown in FIG. 8.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

The present disclosure has been described above with reference toexemplary embodiments. However, those skilled in the art, having readthis disclosure, will recognize that changes and modifications may bemade to the exemplary embodiments without departing from the scope ofthe present disclosure. The changes or modifications are intended to beincluded within the scope of the present disclosure, as expressed in thefollowing claims.

1-19. (canceled)
 20. A method of treating cancer comprising initiatingromidepsin therapy to a cancer patient, wherein said cancer patient hasan expression level of Testis-Specific Y-encoded-like protein 5 (TSPYL5)lower than a defined TSPYL5 expression level threshold.
 21. The methodof claim 20, wherein the level of the TSPYL5 expression is determined bymeasuring the amount of TSPYL5 protein using an immunoassay.
 22. Themethod of claim 21, wherein the immunoassay is an immune-polymerasechain reaction (immuno-PCR).
 23. The method of claim 20, whereinromidepsin is administered intravenously.
 24. The method of claim 23,wherein the dose of romidepsin is a range of between 0.5 and 28 mg/m².25. The method of claim 24, wherein the dose of romidepsin is a range ofbetween 8 and 14 mg/m².
 26. The method of claim 25, wherein the dose ofromidepsin is about 8 mg/m².
 27. The method of claim 25, wherein thedose of romidepsin is about 10 mg/m².
 28. The method of claim 25,wherein the dose of romidepsin is about 12 mg/m².
 29. The method ofclaim 25, wherein the dose of romidepsin is about 14 mg/m².
 30. Themethod of claim 25, wherein romidespin is administered in the dose ofabout 14 mg/m² an as IV infusion over a 4 hour period on days 1, 8, and15 of the 28 day cycle.
 31. The method of claim 20, wherein romidepsinis administered orally.
 32. The method of claim 31, wherein the dose ofromidepsin is a range of between 10 and 300 mg/m².
 33. The method ofclaim 32, wherein the dose of romidepsin is a range of between 25 and 75mg/m².
 34. The method of claim 33, wherein the dose of romidepsin isabout 25 mg/m².
 35. The method of claim 33, wherein the dose ofromidepsin is about 50 mg/m².
 36. The method of claim 33, wherein thedose of romidepsin is about 75 mg/m².
 37. The method of claim 33,wherein romidespin is administered in the dose of about 50 mg/m² on days1, 8, and 15 of the 28 day cycle.
 38. The method of claim 20, whereinthe cancer is leukemia or lymphoma.
 39. The method of claim 20, whereinthe cancer is a solid tumor selected from the group consisting of lung,breast, colon, liver, pancreas, renal, prostate, ovarian and braincancer.